Abstract

Neutrophils are recruited into the female reproductive tract following insemination to eliminate excess spermatozoa and bacteria introduced by the breeding process. In natural breeding, bovine semen is deposited in the vagina and sperm migrate into the uterus, leaving the bulk of seminal plasma (SP) behind. Current artificial insemination protocols introduce variable amounts of SP and semen extender into the uterus. Beside phagozytosis and secretion of immune modulators, polymorphonuclear neutrophils (PMN) are able to form neutrophil extracellular traps (NETs) extruding their DNA into the extracellular environment and ensnare pathogens including sperm cells. Recently, a time-dependent increase in bovine NETs formation has been reported. In the absence of SP and extender only a low degree of NETs formation by the sperm cells alone was detected (Fichtner et al. 2017 Reprod. Domest. Anim. 52(S1), 16). The aim of the present study was to investigate the effect of different semen extenders with various supplements or varying SP concentrations on NETs formation. Bovine PMN were isolated via Ficoll gradient centrifugation from peripheral blood. Semen extenders, purchased from 2 companies, were supplemented either with no animal protein or fresh egg yolk or “egg yolk-like substances” (gamma-irradiated sterile egg yolk or phospholipids as liposomes). The SP collected from ejaculates of eight 5-year-old bulls was added to the incubation medium in concentrations of 1, 3, 5, 10, 15, and 20%. For NETs induction, PMN and semen extenders or SP concentrations were co-cultured for 60 min; PMN and incubation medium alone served as negative controls. After incubation, the samples were treated with micrococcal nuclease and stained with PicoGreen. Quantification was performed by spectrofluorometric analyses using an automated plate monochrome reader (Varioscan Flash; Thermo Scientific, Waltham, MA, USA). Relative fluorescence intensities (FI in arbitrary units, AU) were calculated by subtracting the values of the negative controls from the ones obtained from the different samples. For statistical analyses, a one-way ANOVA followed by Tukey’s HSD test was used. Experiments were repeated at least 9 times. No significant differences in the relative FI were detected between the 2 animal protein-free extenders. A significantly higher FI was observed in one of the extenders supplemented with egg yolk compared with the other. The same held true for one extender completed with an egg yolk-like substance. Relative FI significantly increased from 1 to 5% SP, stayed constant up to 10%, followed by a slight decrease up to a concentration of 20%. These data indicate that formation of NETs in vitro depends on the composition of the extender itself and the protein source used by different companies. Furthermore, formation of NETs is also dependent on the dose of SP. From these results, it could be speculated that semen extenders plus additives and SP may affect fertility.

The financial support of the Förderverein Bioökonomieforschung e.V. (FBF) is gratefully acknowledged.